Objectives: To purify and determine the amino acid sequence of cytochrome c1, and to study the evolution of electron transfer proteins in general. Cytochromes c1's role in energy production (e.g., the heart) via oxidative phosphorylation, its possible role in divalent cation transport (Ca ions is central to nervous tissue function), and its interaction via electron transfer with cytochrome c, whose evolutionary history is known from bacteria to man in over 40 species, form the scientific basis for these studies. The preparation of high-purity cytochrome c1 has been simplified by preparative gel isotochophoresis as the final purification step. The isoelectric point of cytochrome c1 has been determined to be approximately 4.2, suggesting its very acidic character. This has been comfirmed by amino acid analysis. This acidic character is in marked contrast to cytochrome c which is a basic protein with pI equal to 10.7, and to which cytochrome c donates electrons. Our stochastic model of protein evolution has been extended to predict a priori the number of nucleotide differences between each of 741 pairs of cytochrome c cistrons. This prediction permits an unambiguous discrimination as to which of the two current models of protein evolution is correct.